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Notes: Expression of SerpinB2 by by activated monocytes and macrophages is up-regulated during inflammatory processes and following infection with certain parasitic, viral and bacterial pathogens. These authors identified SerpinB2 as a potentially important host factor in enhancing HIV transcription. They showed that HIV-1 infection and gp120 treatment of peripheral blood mononuclear cells caused induction of SerpinB2, and that SerpinB2 expression resulted in enhanced viral replication. Viral transcription was increased 3-10 fold in cells expressing SerpinB2 and was reduced in macrophages from SerpinB2 knockout mice. They used a series of truncated HIV-1 promoter constructs to localize the region associated with SerpinB2 enhancement of transcription to a region of the HIV-1 long terminal repeat promoter containing three Sp1 binding sites. They used luciferase reporter constructs and beta-galactosidase control vectors in these reporter assays. (3710)

Notes: The researchers sought to determine whether PPARγ expression might direct the effects of gastrin in proliferation of colorectal cancer cells (CRC). They determined that cell line DLD-1 cells had both PPARγ and gastrin receptors. They demonstrated that gastrin stimulated CRC cell proliferation with a coincident decrease in PPARγ levels. These studies show that gastrins trophic properties could be due in part to transactivation of EGFR and phosphorylation of ERK1/2, leading to a decrease in PPARγ activation.

The authors used CellTiter 96® Aqueous One Solution Cell Proliferation Assay to measure cell growth of the CRC cell line DLD-1.

The DLD-1 cells were transiently transfected with a luciferase vector, and FuGENE® 6 Transfection Reagent was used at a DNA ratio of 3:1 in 24-well plates. To normalize for transfection efficiency, the cells were co-transfected with a β-Gal reporter construct. The Dual-Luciferase® Assay System was used to measure PPARgamma activity with values then normalized to Beta-Gal, measured with the β-Galactosidase Enzyme Assay System. (4280)

Notes: Total RNA was isolated from TOP10F’ or INVaF' E. coli (Invitrogen) using the SV Total RNA Isolation System. The isolated RNA was used in RT-PCR to detect cleavage of marA mRNA. A marA drug resistance gene sequence was also ligated into a chloramphenicol acetyltransferase (CAT) reporter vector. This construct was used to measure single-stranded nanovector-transcribed ribozyme activity on the marA sequence. CAT activity was measured using the CAT Enzyme Assay System. CAT activity was normalized by cotransfecting cells with the pSV-β-Galactosidase Vector and assaying lysates with the β-Galactosidase Enzyme Assay System. (2794)

Notes: Most organisms display an endogenous timekeeping mechanism, or circadian clock, which consists of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness. In Drosophila, as well as other organisms, several of the molecules involved in sustaining this circadian clock have been identified. A gene product required for circadian photoreception has recently been identified in Drosophila, and termed crytochrome (CRY). These researchers investigated whether CRY could interact directly with the core clock proteins PERIOD (per) and TIMELESS (tim). The Drosophila cell line S2 was transiently transfected with a firefly luciferase reporter construct under control of the tim promoter, in conjunction with different combinations of constructs expressing clk, per, tim, and cry. Data was normalized to a cotransfected reporter plasmid, either the pRL-null Vector, a Renilla luciferase vector, or a beta-galactosidase expression vector, and the resulting activities measured using either the Dual-Luciferase® Reporter Assay System or the Beta-Galactosidase Enzyme Assay System. These transfection studies, along with coimmuneprecipitation assays, a yeast two-hybrid assay, and immunolocalization studies, show that CRY can block the function of PER/TIM heterodimeric complexes in a light-dependent fashion. In addition, PER/TIM and CRY influence the subcellular distribution of these protein complexes. Thus, CRY acts as a circadian photoreceptor by directly interacting with the core protein components of the circadian clock. (1354)

Notes: Reporter constructs were prepared in the pGL2 Basic Vector and transfected with a beta-galactosidase vector as a control. Transfections into 18–81, 18–8, M12.4.1, A20, Nama-Iwa and Raji B-cell lines was accomplished with the standard ProFection® Mammalian Transfection System-DEAE Dextran. A lot of detail is provided. The DEAE-Dextran from the kit was also used for a DEAE-Dextran electroporation protocol to transfect for two other B cell lines. A lot of detail is provided. Reporter activities in the transfected cells were determined with the Luciferase Assay System and the Beta-Galactosidase Enzyme Assay System. (0566)

Notes: pSV-β-Galactosidase Control Vector was cotransfected as an internal standard. Cell extracts were made 24 hours after the transfection using the Reporter Lysis Buffer and assayed using the β-Galactosidase Enzyme Assay System . (1056)

Notes: The authors used the Primer Extension System to map the transcriptional start site of human CCR5 gene. They cloned the putative CCR5 promoter, and regions thereof were cloned into the CAT in pCAT®-Basic Vector. Transient transfections were controlled for using a β-galactosidase expression plasmid, and β-galactosidase levels assayed using the β-Galactosidase Assay System. The pCAT® Vectors have been replaced by the next generation pCAT®3 Vectors. (1085)

Notes: Cytotoxicity of AT-61 for HepG2 cells an HEPAD38 cells (a HepG2 cell loine that produces HBV when grown in the absence of tetracycline) was assessed using the Cell Titer 96® Non-Radioactive Cell Proliferation Assay. To assay for the inhibition of the tetracycline promoter activity in HepAD43 cells (produces β-galactosidase in the absence of tetracycline), β-galactosidase activity was determined using the Reporter Lysis Buffer from the β-Galactosidase Assay System. (2506)

Notes: A reporter construct was assembled in the pGL3 Control Vector downstream of the Xba I consisting of the 3'UTR of the COX-2 gene. The construct was designed to see if the UTR had an affect on mRNA stability in the presence of IL-1beta. The construct as well as the pSV-Beta Galactosidase Control Vector were transfected into A549 cells using the Tfx™-50 Reagent. A lot of detail is provided for the transfection. The reporter activities were monitored with the Luciferase Assay System and the Beta-Galactosidase Enzyme Assay System. (0606)

Notes: Luciferase (prepared in pGL2-Basic Vector) and β-galactosidase (pSV-β-Galactosidase Control Vector) reporters were studied in SL2 Drosophila cells, HeLa cells and primary rat cortical neurons. Transfections into the SL2 and HeLa cells were accomplished with standard calcium phosphate methods. Transfection of primary rat cortical neurons was accomplished with the Tfx™-50 Reagent as described in Boeckman, F.A. et al. (1996) Neural Notes II(1), 13-15. Reporter assays were performed with the Luciferase Assay System and the β-Galactosidase Assay System. (0890)

Notes: Authors used the Wizard®Plus Megapreps DNA Purification System to isolate DNA and transfect it into GH cells. They also used the β-Galactosidase Enzyme Assay System and Riboprobe® in vitro Transcription Systems in their studies. (0945)

J. Biol. Chem.272, 12692-12698..
Cloning and characterization of the promoter region of a gene encoding a 67-kDa glycoprotein.1997

Chatterjee, N. , Zou, C. , Osterman, J. C. , Gupta, N. K.

Notes: Luciferase studies were performed in KRC-7 rat hepatoma cells using constructs prepared in the pGL3 Basic Vector. The luciferase constructs were cotransfected with the pSV-beta-galactosidase Vector. Luciferase activities determined with the Luciferase Assay System were normalized to readings from the Beta-Galactosidase Enzyme Assay System. (1364)

J. Biol. Chem.272, 5915-5920.
Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes.1997

Price, D. J., Rivnay, B., Fu, Y., Jiang, S., Avraham, S., Avraham, H.

Notes: The pSV-β-Galactosidase Vector was used as transfection control. The protein of interest was detected by western blotting. The amount of protein loaded on the gel was normalized to β-galactosidase activity. (0547)

Notes: Reporter studies were performed in the mouse macrophage-like cell line RAW 264.7. Promoter constructs were prepared in the pGL2 Basic Vector and luciferase activity monitored with the Luciferase Assay System. Transfections were normalized by co-transfecting the pSV-β-Galactosidase Control Vector. The β-gal activity was assayed with the β-Galactosidase Assay System. (0713)

Notes: A 318bp promoter region from genomic DNA was amplified by PCR and ligated into the pGEM®-T Vector. This promoter region was then cloned into the pGL2-Basic Vector, and activity was measured using the Luciferase Assay System. Transfection efficiency was monitored with the β-Galactosidase Enzyme Assay System. (0300)

Notes: Promega's pCAT® Basic and pSV Beta-Galactosidase Vectors were used in this study. Beta-galactosidase activity was assayed with the Beta-Galactosidase Enzyme Assay System with Reporter Lysis Buffer. Promega's pCAT Vectors have now been replaced by the next generation pCAT® 3 Reporter Vectors. (0434)

Notes: Luciferase studies were performed in normal human dermal fibroblasts and primary human astrocytes. The experimental constructs were prepared in the pGL2-Basic and pGL2-Promoter Vectors. All luciferase activities (determined with the Luciferase Assay System) were normalized to β-Galactosidase activity. (0884)

Notes: Luciferase studies were performed in HepG2 cells using the Luciferase Assay System. Experimental constructs were prepared in the pGL2-Basic Vector. Transfection efficiency was standardized using the β-Galactosidase Assay System with Reporter Lysis Buffer. (2245)

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